Vol. 140, No. 3
JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 955-963
0021-9193/79/12-0955/09$02.00/0
Escherichia coli Mutants Tolerant to Beta-Lactam Antibiotics KAZUAKI KITANO AND ALEXANDER TOMASZ* The Rockefeller University, New York, New York 10021 Received for publication 20 July 1979
Two types of Escherichia coli mutants tolerant to beta-lactam antibiotics were isolated. One is E. coli X2452, which showed a tolerant response against betalactam antibiotics when grown at 420C, and the others are the mutants C-80 and C-254, selected from mutagenized E. coli X 1776 by cycles of exposure to ampicillin, cephaloridine, and starvation of the nutritionally required diaminopimelic acid. Beta-lactam antibiotics caused rapid loss of viability and lysis in cultures of X1776 or in X2452 grown at 320C. In contrast, the same antibiotics caused only a reversible inhibition of growth in mutants C-80 and C-254 or in cultures of X2452 grown at 420C. Beta-lactam antibiotics that show high affinity for penicillinbinding proteins 2 or 3 (mecilhinam and cephalexin, respectively) induced similar morphological effects (ovoid cell formation and filament formation) in both parent and mutant strains. In contrast, beta-lactam antibiotics which have a high affinity for penicillin-binding protein 1 (e.g., cephaloridine or cefoxitin), which cause rapid lysis in the parental strains, caused cell elongation in the tolerant bacteria. In contrast to the parental cells, autolytic cell wall degradation was not triggered by beta-lactam treatment of X2452 cells grown at 420C or in mutants C-80 and C254. The total autolytic activity of mutants C-80 and C-254 was less than 30% that of the parent strain. However, virtually identical autolytic activities were found in cells of X2452 grown either at 42 or 320C. Possible mechanisms for the penicillin tolerance of E. coli are considered on the basis of these findings. Several types of biochemical observations suggest that lysis of Escherichia coli by beta-lactam antibiotics involves the activity of autolytic enzymes (5, 18, 19). Evidence for such an involvement exists in pneumococci since mutants selected for a defective autolytic system were found to be resistant to the lytic (and, to some degree, to the bactericidal) effect of beta-lactam antibiotics, while remaining sensitive to the growth inhibitory effect of these antibiotics (25, 26). Repeated attempts to select for autolysindefective E. coli have failed so far. Therefore, we decided to select for penicillin-tolerant mutants of E. coli, i.e., bacteria that could survive treatment with penicillin in a manner that is characteristic of the penicillin-tolerant pneumococci. We describe here three tolerant E. coli isolates. Each mutant was found to have a defective autolytic system. We chose the E. coli K-12 strain X1776 as the parental strain in the mutant isolation because of the presence of several useful properties of which of particular importance were the requirement for diaminopimelic acid (DAP), defective outer membrane, and sensitivity to detergentinduced lysis (4).
Although a complete characterization of the genetic and biochemical basis of tolerance in these bacteria is not yet available, it was felt that the existence of the first penicillin-tolerant, gram-negative mutants was of sufficient interest to warrant a preliminary description. MATERIALS AND MErTODS Bacterial strains and culture conditions. E. coli X2452 F- dapD AlacZ39 A(galbchl) A- tyrT58 naLA29 AthyA57 IyaA32 endAl asd hsdS3 (4), E. coli X1776
F- tonA53 dapD8 minAl supE42 A(gal-uvrB)40 AminB2 rfb-2 nalA25 oms-2 thyA57 metC65 oms-1 A(bioH-asd)29 cycB2 cycAl hsdR2, and E. coli C-80 and C-254 (beta-lactam tolerant mutants derived form X1776) were used. Because of the known mechanical fragility of both strains X1776 and x2452, bacteria were cultured without aeration. Cultures of 10 ml each were grown in test tubes (18 by 150 mm) at 32 or 420C in Penassay broth (Difco antibiotic medium no. 3) supplemented with DAP (20 ,ug/ml), L-lysine (100 yig/ml), biotin (0.2 ,ug/ml), and thymidine (30,ug/ml) (complete medium, OM). Cells radioactively labeled in the cell wall with DAP were obtained by growth in medium in which the DAP was replaced with 1 uCi of [3H]DAP per ml (giving a final DAP concentration of 3.1 ,ug/ ml). Isolation of mutants. E. coli X1776 was mutagen-
955
956
KITANO AND TOMASZ
ized by conventional procedures (1). Bacteria, grown exponentially at 320C in complete medium, were suspended in 0.05 M Tris-maleate buffer (pH 6.0) containing 300 ,ug of N-methyl-N'-nitro-N-nitrosoguanidine and were incubated for 30 min at 37°C. Celis were washed once with Tris-maleate buffer to remove the mutagen and suspended in OM. Portions of 2 ml were inoculated into 8 ml of OM and incubated at 320C for 3.5 h (cell concentration at this point was 5 x 107 to 7 x 107 cells per ml). Mutants were selected next by a cycle of treatments that included the following steps. Step (i). A 10-ml mutagenized culture was treated with ampicillin (20 ug/ml, corresponding to about five times the minimal inhibitory concentration [MIC]) for 1.2 h. During this treatment, the turbidity of the bacterial culture first increased by about 30% and then dropped to about 50% of the maximum turbidity value. The cells were collected by centrifugation (5,000 x g, 10 min, room temperature), washed once with OM, and resuspended in fresh OM (twice the original volume). After overnight incubation (13 h) at 320C, the surviving cells grew to a turbid culture that was resuspended in fresh OM and allowed to double in its turbidity (5 x 107 to 10 x 107 per ml), at which time a 10-ml portion of the culture received step (ii) of the enrichment procedure. Step (ii). Step (ii) consisted of treatment with cephaloridine (20 jug/ml; five times the MIC value) at 320C for 1.5 h. The turbidity changes of this culture were about the same as those observed during the first ampicillin treatment. After the cells were washed with OM, dilution and overnight incubation were the same as after ampicillin treatment. The overnight culture was washed with OM without DAP, resuspended in OM without DAP, and incubated at 320C for 3 h. Step (iii). During this DAP starvation there was an initial 20% increase followed by a decline (about 30% of the maximum) in turbidity. The culture was washed and resuspended in OM (to half of the culture's turbidity at the end of the DAP starvation) and incubated (320C) for 3 h, during which time the turbidity doubled. Portions of 10 ml of this culture were then exposed to the final step. Step (iv). Step (iv) consisted of ampicillin treatment (20 ,g/ml for 2.5 h). The bacteria were finally washed with OM and resuspended in 10 ml of OM, and frozen stocks were prepared (freezing at -40°C and storage at -80°C). The final scoring of mutants was done in the following way: frozen stock cultures were melted, diluted, and plated on OM agar plates (about 107 bacteria per plate) containing 10 Ag of ampicillin per ml and incubated at 320C for 16 h. Then the plates were overlayered with 4 ml of soft agar medium (0.6% agar in OM medium) containing 10 U of penicillinase (Rikers Chemicals) per ml and incubated for another 24 h. The colonies that grew up under this condition (about 100 per plate) were picked into liquid medium and checked for a tolerant response against beta-lactam antibiotics. Out of 340 such surviving colonies, 6 were found to show a substantially decreased rate of loss of turbidity (as compared with the parental culture) when challenged with cephaloridine (five times the MIC). These cultures also showed lower autolytic activity (30 to 60% of the parental cells). Two of these
J. BACTERIOL.
tolerant mutants, C-80 and C-254, exhibiting virtually no lysis during cephaloridine treatment, were chosen for more detailed characterization to be described in this paper. Assay procedures. Bacteria were routinely grown at 320C except when noted otherwise. Culture growth and culture lysis were monitored with a Coleman Nepho-colorimeter (14). Viable titers of the cultures were assayed by routine plating procedures. The assay method of triggered autolysin by beta-lactam antibiotics (13) was as follows. After growth in medium supplemented with [3H]DAP for several generations, cells in the exponential growth phase were collected by centrifugation (4,300 x g, 5 min), transferred to radioactive isotope-free growth medium, and incubated for 50 min (i.e., a period of about one generation) to deplete cellular pools of the [3H]DAP. After this period, 1.5-ml portions of the culture were distributed into a number of small tubes containing beta-lactam antibiotics at various concentrations (representing multiples of the corresponding MICs) and incubated for an additional 10 to 20 min. The cultures were chilled (ice bath) and then centrifuged at 3,300 x g for 5 min at 40C. Cells were washed with 1.5 ml of ice-cold phosphate buffer (0.1 M, pH 7.0), resuspended in 1.5 ml of the same buffer containing 10 mM MgSO4, and incubated at 320C. The total time needed to transfer the cells to the buffer was about 10 to 15 min. After 0, 30, 60, 90, and 120 min of incubation, 200-p1 portions were removed into prechilled Eppendorf microcentrifuge tubes containing 20 pl of 38% formaldehyde (to stop murein hydrolase activity). After centrifugation at 12,000 x g for 10 min in the cold (40C), radioactivity in 100 pl of the supernatant fluids was counted. To determine total radioactivity of the reaction mixture, 200-pd portions were mixed with 20 p1 of 4% deoxycholate and incubated for 30 min at 320C; a 100-pl portion of this suspension was used to determine radioactivity. The activity of autolysin triggered by beta-lactam antibiotics was expressed as the rate of degradation of murein during a 2-h incubation of the beta-lactam antibiotic-treated cells in buffer. The rates were corrected for the spontaneous rate of release of radioactivity from the control (untreated) cells. The treatment time with beta-lactam antibiotics was 20 min (for E. coli X2452 cells) or 10 min (for the cells of E. coli x1776 C-80 and C-254). Triggering of murein hydrolase by trichloroacetic acid. The method of Schwarz et al. was used (18) (see also legend to Fig. 8) to trigger murein hydrolase by trichloroacetic acid. Reagents. DL-(meso)-2,6-DiamiIno-[U-3H]pimelic acid (1.5 Ci/mmol) was purchased from Amersham Corp. (Arlington Heights, Ill.). All other materials and chemicals were reagent-grade commercially available products.
RESULTS Tolerant response of E. coli X2462 to betalactam antibiotics at 420C. Several mutants of E. coli K-12 were examined for their response to beta-lactam antibiotics and a mutant, E. coli X2452, was found to show a tolerant response to
BETA-LACTAM-TOLERANT MUTANTS OF E. COLI
VOL. 140, 1979
beta-lactams at 42°C but not at 32°C (Fig. 1). X2452 was isolated by Dennis Pereira in the laboratory of Roy Curtiss (D. Pereira, Ph.D. Thesis, University of Alabama Medical Center, Birmingham, Ala., 1979). Although rapid culture lysis occurred after exposure to the MIC of cephaloridine at 320C, the cells were stable to lysis by the addition of cephaloridine when the bacteria were grown at 420C. This phenomenon was reproducible by the addition of other betalactams such as ampicillin, benzylpenicillin, or cephalothin. The MICs of these beta-lactams were identical at the two temperatures (Table 1). The tolerant response to beta-lactam antibiotics was temperature dependent, and if temperature was shifted down from 42 to 320C at 0 to 30 min after the addition of the drug, cells started lysing after a short time lag (Fig. 2). Tolerant response of E. coli C-80 and C254 to beta-lactam antibiotics. Figure 3 demonstrates the effect of cephaloridine on cultures of E. coli X1776, C-80, and C-254. Although growth of the mutants was stopped by the addition of cephaloridine, the cells scarcely lysed even after prolonged (overnight) incubation with the antibiotic. Culture lysis was very slow or negligible in all of the tolerant mutants (including X2452 grown at 4200) even during exposure
957
TABLE 1. MICs of beta-lactam antibiotics against mutants of E. colia MIC (jug/ml) of strain: Antibiotic
X2452
X1776
C-80
C-254
320C 420C 3.9 3.9 3.9 3.9 15.6 15.6 31.25 31.25
Cephaloridine 2.0 3.9 3.9 Ampicillin 3.9 3.9 3.9 Cephalothin 31.25 62.5 62.5 Benzylpenicillin 31.25 62.5 62.5 Cefoxitin 7.8 7.8 7.8 Cephalexin 15.6 Mecillinam 7.8 7.8 7.8 aThe MICs listed in the table were determined by the following procedure: exponentially growing cultures (at cell concentration of about 5 x 107 cells per ml) received antibiotics at various concentrations, and growth of the cultures was monitored (as described in the Materials and Methods). The minimum antibiotic concentration that caused cessation of growth was taken as the MIC. Inhibition of growth was not instant but became manifest only after a residual increase in the turbidity of cultures.
400t
N 200}
42 C 100'
50'
0
1
2
HOURS FIG. 2. Effect of temperature shift during treatment of E. coli X2452 with cephaloridine. Bacterial cultures were grown in OM at 42°C without aeration. At a cell concentration of about 1 x io0 viable cells per ml (in the late exponential phase of growth) (arrow), the cultures received 2.5 times the MIC of cephaloridine (19.5 pg/ml), and the temperature of the cultures was shifted to 320C at 0 (A), 15 (V), and 0 1 2 3 0 1 2 3 30 (El) min after the addition of the drug. Control HOURS cultures at 42°C with (O) or without (*) cephaloriFIG. 1. Effect of cephaloridine on cultures of E. dine are also shown. coli X2452 growing at 32 and 42°C. Bacterial cultures were grown in OM without aeraition. In the late to high concentrations (eight times the MIC) of exponential phase of growth (arrrow), the cultures and a variety of other cephaloridine, received cephaloridine at the conc ples of MIC; 7.8 pg/ml at both te,mperatures) indi beta-lactam antibiotics. Nevertheless, inhibitors cated by the numbers, and the grou pth response of the of early stages of cell wall synthesis (e.g., the bacterial cultures was followed by inephelometry and combination of D-cycloserine and fluoro-D-alaplotted in nephelometric (N) units. nine) could still induce lysis of the tolerant bac-
ampicillin,
958
KITANO AND TOMASZ
J. BACTERIOL.
X-1 776
C-254
C -80
400
N
1004 so
0
1
2
3
0 1 2 3 0 1 2 3 HOURS FIG. 3. Effect of cephaloridine on cultures of E. coli X1776 and its mutants C-80 and C-254. Bacterial
cultures were grown in OM at 32°C without aeration. In the exponential phase ofgrowth (5 x 107 viable cells per ml) (arrow), the cultures received antibiotics at the concentrations (multiples of MIC) indicated by the numbers, and the growth response of the bacterial cultures was followed by nephelometry. The MICs for cephaloridine were as follows: xl776, 3.9 pg/ml; mutants C254 and C-80, 7.8 pg/ml.
teria (data not shown). The MICs of various beta-lactams (for method of MIC determination see footnote a, Table 1) were identical to those of the parental cells (Table 1) except for benzylpenicilhin and cephalothin; in these cases the MIC for the tolerant cells was higher (maximum, twice those of the parents). Cultivation of E. coli X2452 at 420C gave a striking protection against the killing effects of cephaloridine (Fig. 4a) and also against lysis. The killing effects of cephaloridine in the mutants C-80 and C-254 were also very weak as compared with the effects in the parental strain (Fig. 4b). Morphological changes of the mutant cells during treatment with beta-lactams. Figure 5 demonstrates the effect of three betalactam antibiotics on the cell shape of E. coli X2452 at 42 and 320C. Cephaloridine, which has a high affinity for penicillin-binding protein (PBP) lb (20, 21) caused rapid lysis of cells at 320C and the formation of empty cells, whereas at the nonpermissive temperature for lysis, cell elongation continued, and bacilli became noticeably longer than the control cells. Mecillinam (which has a high affinity for PBP 2 [20]) caused the formation of ovoid cells at both 32 and 420C. During the 3 h of treatment with cephalexin, cells grown at 420C have elongated to lengths varying between 1.5 and 3 times the length of the untreated bacteria grown at this temperature. The same drug caused a more substantial elongation (3 to 6 times the normal length) when
CX-254
x
C°~X-1776
4._ co
B 2
1
2
HOURS FIG. 4. Bactericidal action of cephaloridine against beta-lactam-tolerant mutants of E. coli. (A) E. coli X2452 cells growing in complete medium at 32 and 42°C were treated at 0 min (cell concentration, 1 x 108 bacteria per ml) with 20 pg (about 2.5 times the MIC) of cephaloridine per ml. (B) Cells of E. coli X1776 C-80 and C-254 growing in complete medium at 320C were treated at 0 min with twice the MIC of cephaloridine. Samples were periodically removed, diluted with complete medium, and plated to determine the number of viable cells.
the cells were grown at 320C. Similar phenomena were also observed with the tolerant mutants C-80 (Fig. 6) and C-254 (data not shown). In contrast, the mecillinam-induced ovoid cell formation was not easy to document in these tolerant strains or in the parental strain X1776. Mecillinam treatment, at least under the conditions used here (i.e., relatively high cell concen-
VOL. 140, 1979
vi. .i,j4
~*
959
BETA-LACTAM-TOLERANT MUTANTS OF E. COLI -4
........ . . t~ ~ ~ ~ ~ ~ ~ ~ . .
.jt
*$,. .. . b
I! \
:~~~~~ *_
A.
(
B~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~:
4~~. 2.B i-
1A¢.-: . x At; .:....''
v
4
.
S'M |
..
/
:0
. ,f '
.d'':
.I
dp
I
/_....
IF
^
6
I
_1 lb
I-
_a
IN
.FIG. 5. Effect of beta-lactam antibiotics on cell shape of E. coli X2452 grown at 32 and 42°C. (A) Control; (B) cephaloridine; (C) mecillinam; (D) cephalexin. Cells grown at 32 and 42°C were exposed to four times the MICs of various beta-lactam antibiotics for 3 h. Cells were fixed with 2% glutaraldehyde, and phase-contrast micrographs were then made of the cells, using a Zeiss microscope fitted with a Planachromat 100/1.25 phasecontrast oil immersion objective. Kodak Panatomic-X film was used for the photomicrography.
tration), caused the formation of abnormally shaped bacteria (Fig. 6). Triggering of autolytic activity by cephaloridine and by trichloroacetic acid treatment. The autolytic cell wall degradation of E. coli X2452 was effectively triggered by brief exposure (10 to 20 min) to beta-lactam antibiotics at the lysis-permissive temperature but was not triggered at 42°C (Fig. 7). Autolysis of mutants C-80 and C-254 was not triggered by this treatment either (Fig. 7). Interestingly, exposure to high concentrations of antibiotics (eight times the MIC [Fig. 7]) has occasionally caused a suppression of the rate of cell wall degradation. This type of effect has been consistently observed at very high concentrations (10 to 100 times the MICs) of certain beta-lactam antibiotics (13). The autolytic activity of E. coli X2452 triggered by 5% trichloroacetic acid treatment (7) was almost the same in cells grown either at 32 or 420C and was independent of the temperature at which the assays were carried out (Fig. 8a). In contrast, the autolytic activities of C-80 and C-254 triggered by 5% trichloroacetic acid treatment were less than 30% that of the parent strain
(Fig. 8b). Identical results were obtained when autolysis was triggered by other methods (exposure to hypertonic sucrose or to freezing and thawing [7]) or in experiments in which the autolytic activities of Triton-ethylenediaminetetraacetic acid extracts (7) (made from tolerant and lysis-prone cells) were compared. DISCUSSION The mechanisms by which inhibition of penicillin-sensitive bacterial enzymes (or PBP) brings about interference with the growth of a bacterial cell is not clearly understood presently (24). Chemically different beta-lactam antibiotics can elicit a variety of different morphological effects and distinct growth inhibitory mechanisms in the same bacterium (E. coli) (20, 21). There are also examples of the converse situation since different species of bacteria treated with the same beta-lactam antibiotic may show a wide variety of physiological responses, such as inhibition of growth (with only an extremely slow loss of viability), rapid loss of viability, and loss of viability accompanied by cellular lysis (9, 26). Studies on pneumococci and some other gram-positive bacteria have shown that the
960
KITANO AND TOMASZ
X -17 76
;
|
J. BACTERIOL.
_
........
000, .:....::.
C-80
4koolo
q*4
0 4e : :. .,.1
a 0
I
Ls
A,
I
0
/
v
1.
FIG. 6. Effect of beta-lactam antibiotics on cell shape of E. coli X1776 and its mutants. (A) Control; (B) cephaloridine; (C) mecillinam; (D) cephalexin. Cultures of the bacteria grown at 32°C were exposed to four times the MICs of the various beta-lactam antibiotics for 3 h. After fixation with 2% glutaraldehyde, phasecontrast micrographs were made of the bacteria, using a Zeiss microscope fitted with a Planachromat 100/ 1.25 phase-contrast oil immersion objective. Kodak Panatomic-X filn was used for the photomicrography.
beta-lactam antibiotic-induced lysis requires the activity of bacterial autolysins (2, 6, 26). Pneumococci in which the in vivo activity of the Nacetyl muramic acid-L-alanine amidase has been suppressed (by mutation or physiological manipulations) show a unique response to penicillin treatment: in such cells the antibiotic causes inhibition of growth (at the normal MIC) and often the rate of loss of viability is also greatly reduced, but no lysis occurs (25, 26). We proposed the term "antibiotic tolerance" for this phenomenon (25), and recent reports suggest that antibiotic tolerance may not be restricted to laboratory strains of pneumococci but may also occur among clinical isolates of Staphylococcus aureus (17) and Streptococcus sanguis (10). The involvement of autolytic activity in the penicillin-induced lysis of E. coli has been repeatedly suggested in the literature, and penicillin treatment of E. coli has been reported to cause enzymatic cell wall degradation (19) and also an increase in the in situ autolytic activity (5). In a recent, more detailed study, we found a striking quantitative correlation between the efficiency of beta-lactam antibiotics to trigger autolytic cell wall degradation and the relative affinity of these antibiotics for the PBP 1 group of E. coli (13). In this com-
munication we describe the properties of three E. coli isolates that exhibit a tolerant response to treatment with penicillins and cephalosporins. Each one of the isolates has a defective autolytic system. One of the isolates (x2452) exhibits a tolerant response when grown at 420C and a lytic bactericidal response when grown at 32°C. The other two mutants (C-80 and C-254) show tolerance at all temperatures of growth. These E. coli mutants resemble the autolysis-defective pneumococci in that each one of them exhibits an abnormality in their autolytic system: brief treatment of the mutants (or of X2452 grown at 4200) with beta-lactam antibiotics does not trigger cell wall degradation, although the same treatment causes rapid and extensive wall hydrolysis in the parental bacteria and in strain X2452 grown at the lysis-permissive temperature (320C). Additional tests have revealed a substantially decreased autolytic activity in the mutant cells (as compared with the parental bacteria). However, cells of strain X2452 grown at either 32 or 420C appear to have comparable (normal) levels of autolysin activity. Thus, tolerance of mutants C80 and C-254 is accompanied by an apparent net decrease in autolysin activity, whereas in X2452 only the triggering of autolytic activity seems
BETA-LACTAM-TOLERANT MUTANTS OF E. COLI
VOL. 140, 1979
961
The mechanism by which interference with cell wall synthesis provokes autolytic cell wall 32 20D degradation in E. coli is not yet known. In the .0 0 case of pneumococci, we proposed that the suia2.0~ cidal activity of the autolytic amidase may be caused by a defect that rapidly upsets the in control of this enzyme after the addition of vivo , 1.5 to the bacteria (26). This suggestion penicillin 0 on the demonstrated release of an was based 30 autolysin inhibitory agent (Forssman antigen) ._ 31 42 C 1.0D from pneumococci during penicillin treatment (26). There is no evidence at this time for the existence of analogous autolysin inhibitors in E. 2 4 8 1 coli. The triggering of in situ autolytic wall degAntibiotic concentration (MIC units) radation of E. coli after exposure of the cells to FIG. 7. Triggering of autolytic activity by cepha- various chemical treatments (e.g., cold trichloloridine. After growth in medium supplemented with roacetic acid, ethylenediaminetetraacetic acid, [3H]DAP for several generations, cells were grown in radioisotope-free medium for an additional cell gen- high concentration of sucrose [7]) or mechanical eration. After this period, 1.5-ml portions of the cul- disruption has been explained as the disruption ture were treated with increasing concentrations of of a "barrier" (plasma membrane?) that was cephaloridine for 20 min (strain x2452) or for 10 min suggested to separate the autolysins of E. coli (strain xl776 and its mutant derivatives). The growth from their substrate in the normal cells (7). temperature was 320C for all strains except for a Rapid release of substantial quantities of lipids portion of X2452, which was also grown at the lysis- and other membrane components have been protective temperature of 42°C. For the assay of cell demonstrated during penicillin treatment of sevwall degradation, cells of all strains (including the 420C-grown bacteria) were incubated at 32°C after eral autolysin-defective gram-positive bacteria washing and resuspension in buffer (1.5 ml). Radio- (9,11, 12). Preliminary experiments indicate that activity released during 2 h of incubation in the buffer penicillin-induced release of lipid material also occurs in some strains of E. coli (A. Tomasz, was counted, and the autolytic activity was expressed unpublished data), and it is conceivable that as the rate relative to the spontaneous rate of release of radioactivity from the untreated bacteria. This such a process may damage the hypothetical X-2452
C
,,, ;
latter value has amounted to about 8% of the total incorporated radioactivity (13).
thermosensitive. The titration of the total autolysin content of E. coli presents considerable problems since there are several enzyme activities involved (8, 16) and some of these are structure bound. Furthermore, in extracts, some of these hydrolytic enzymes may be restricted in their ability to attack cell walls. For these reasons, it was felt that a determination of in situ autolysin activity (7) as it was done in our experiments may be an appropriate first approximation when comparing autolysin levels of tolerant (mutant) and lysis-prone (parental) bacteria. The level of autolysin activity was also determined in Triton-EDTA extracts (7) prepared from the parental cells and from the mutants C80 and C-254. The mutant extracts showed a diminished specific hydrolase activity (about 25 to 30% that of the extract from parent cells). In these assays radioactive DAP-labeled cell walls (murein sacculi [3]) from the parental bacteria were used as substrate (data not shown). It is not yet known whether the lowered autolysin activity in the mutants involves one or more of the multiple types of murein hydrolases of E. coli (8, 16).
60
A
B
.C 50
E 42
X-1776 ,
/
0 3 *
0
;
32 C C ~~~~~~42
.'20
C-254
=>1> ,,
1
,
2
;AtS--~~~~~80 1
2
HOURS FIG. 8. Autolytic activity triggered by trichloroacetic acid treatment. [3H]DAP-labeled cells were suspended in 0.01 M Tris-maleate buffer (pH 6.0) containing 10 mM MgSO4 and mixed with an equal volume of 10% trichloroacetic acid and allowed to react for 10 min in ice (7). The cells were washed three times by centrifugation in the buffer, resuspended in the same buffer, and incubated at 32°C. The radioactivity released into the supernatant solution was assayed at different times by theprocedure described in Materials and Methods. (A) Cells of X2452 grown at 32°C (O) or 420C (*). (B) Cells of X1776 (O) and its mutants C-80 (*) and C-254 (A). Bacteria were grown at 320C.
962
KITANO AND TOMASZ
barrier separating autolysins and the murein in a manner somewhat analogous to the damage that may be responsible for triggering by chemical treatments. If this were true, then the lack of autolysin triggering in strain X2452 grown at 420C may be caused by the presence of a more stable barrier (different membrane composition). Alternatively, cell wall synthesis at the higher temperature may produce a murein of low autolysin sensitivity at a critical area of the mutant cell wall. It should be emphasized that the nature of autolysin triggering is obscure at the present time. There is strong evidence implicating the functional involvement of both the binding proteins 1 (PBP la and lb) (21-23) as well as the autolytic enzyme(s) of E. coli in the penicillininduced lysis of this bacterium. However, it is not clear how and why inhibition of PBP leads to initiation of autolytic cell wall degradation, and the term triggering refers to this poorly understood connection between the functions of PBP 1 and the control of autolysin activity. Besides the hypothetical barrier function, triggering may involve accumulation of cell wall precursors or even the local modification of the murein substrate, e.g., by introduction of poorly cross-linked material into areas of the cell wall during penicillin treatment (15). In the thermosensitive tolerant mutant X2452 the heat-sensitive element in the penicillin response seems to be this hypothetical triggering process since both autolytic activity and the PBP patterns are normal at each temperature (data not shown). The substantially lowered autolytic activity and antibiotic tolerance of mutants C-80 and C-254 are reminiscent of the properties of autolysis-defective pneumococci. On the other hand, lysis of the E. coli mutants can still be induced by treatment with inhibitors of early steps in cell wall biosynthesis, whereas the autolysis defective pneumococci are resistant to the lytic effect of all cell wall inhibitors (25). It is conceivable that the tolerant phenotype has a more complex biochemical and genetic basis in E. coli than in pneumococci because of the more complex nature of autolysin regulation and the differences in the mode of cell wall assembly in E. coli and in pneumococci. The morphological changes induced in the tolerant E. coli during treatment with different beta-lactam antibiotics require comment. It seems that inhibitors of PBP 2 (mecillinam) and PBP 3 (cephalexin) can elicit their typical morphological effects in these cells (i.e., ovoid cell formation and filament formation), whereas the response of the tolerant E. coli to inhibitors of PBP 1 is changed: instead of the appearance of
J. BACTERIOL.
"rabbit ear" forms and lysis, such cells would elongate (Fig. 5). These findings support the notion that in E. coli triggering of autolysin activity by beta-lactam antibiotics is a specific consequence of the inhibition of PBP 1. The tolerant cells appear to be defective in the sequence of events leading from PBP 1 to autolysin triggering; they respond to inhibitors of PBP 1 by reversible inhibition of growth (instead of loss of viability and lysis). These observations suggest that triggered autolysin activity may be the cause of viability loss (as well as cell lysis) in E. coli. It is hoped that these mutants will be helpful in the analysis of the mechanism of the antimicrobial effect of beta-lactam antibiotics. A mutant of E. coli strain K-12 that is tolerant to lysis by cephalexin has been isolated independently by T. Nikaido, S. Tomioka, and M. Matsuhashi (personal communication). No defect in a peptidoglycan-lytic enzyme activity has so far been demonstrated. ACKNOMWLEDGMENTS These investigations have been supported by grants from the National Science Foundation (PCM 7812770) and the National Institutes of Health (AI 12932). We thank Roy Curtiss III and Dennis Pereira (University of Alabama, Birmingham, Ala.) for providing us with strains X1776 and X2452
LITERATURE CITED 1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. Optimal conditions for mutagenesis by N-methyl-N'nitro-N-nitrosoguanidine in Escherichia coli. Biochem. Biophys. Res. Commun. 18:788-795. 2. Ayusawa, D., Y. Yoneda, K. Yamane, and B. Maruo. 1975. Pleiotropic phenomena in autolytic enzyme(s) content, flagellation, and simultaneous hyperproduction of extracellular a-amylase and protease in a BaciUus subtilis mutant. J. Bacteriol. 124:459-469. 3. Braun, V., and K. Rehn. 1969. Chemical characterization, spatial distribution and function of a lipoprotein (murein-lipoprotein) of the E. coli cell wall: the specific effect of trypsin on the membrane structure. Eur. J. Biochem. 10:426-438. 4. Curtiss, R. m. 1978. Biological containment and cloning vector transmissibility. J. Infect. Dis. 137:668-675. 5. Fontana, R., G. Satta, and C. A. Romanzi. 1977. Penicillins activate autolysins extracted from both Escherichia coli and Kkbsiella pneumoniae envelopes. Antimicrob. Agents Chemother. 12:745-747. 6. Forsberg, C., and H. J. Rogers. 1971. Autolytic enzymes in growth of bacteria. Nature (London) 229:272-273. 7. Hartmann, R., B. S. Bock-Henning, and U. Schwarz. 1974. Murein hydrolases in the envelope of Escherichia coli. Eur. J. Biochem. 41:203-208. 8. Holtje, J. V., D. Mirelman, N. Sharon, and U. Schwarz. 1975. Novel type of murein transglycosylase in Escherichia coli. J. Bacteriol. 124:1067-1076. 9. Horne, D., R. Hakenbeck, and A. Tomasz. 1977. Secretion of lipids induced by inhibition of peptidoglycan synthesis in streptococci. J. Bacteriol. 132:704-717. 10. Horne, D., and A. Tomasz. 1977. Tolerant response of
VOL. 140, 1979
BETA-LACTAM-TOLERANT MUTANTS OF E. COLI
Streptococcus sanguis to beta-lactams and other cell wall inhibitors. Antimicrob. Agents Chemother. 11: 888-896. 11. Horne, D., and A. Tomasz. 1979. Release of lipoteichoic acid from Streptococcus sanguis: stimulation of release during penicillin treatment. J. Bacteriol. 137:11801184. 12. Kikuchi, M., T. Kanamaru, and Y. Nakao. 1973. Relation between the extracellular accumulation of L-glutamic acid and the excretion of phospholipids by penicillin-treated Corynebacterium alkanolyticum. Agr. Biol. Chem. 37:2405-2408. 13. Kitano, K., and A. Tomasz. 1979. Triggering of autolytic cell wall degradation in Escherichia coli by beta-lactam antibiotics. Antimicrob. Agents Chemother. 16:838848. 14. Meynell, G. G., and E. Meynell. 1970. Theory and practice in experimental bacteriology. Cambridge University Press, Cambridge. 15. Oka, T. 1976. Mode of action of penicillins in vivo and in vitro in Bacillus megaterium. Antimicrob. Agents Chemother. 10:579-591. 16. Pelzer, H. 1963. Mucopeptidhydrolasen in Escherichia coli 13 I. Nachweis und Wirkungspecifitat. Z. Naturforsch. 18B:950-956. 17. Sabath, L. D., N. Wheeler, M. Laverdiere, D. Blazevic, and B. Wilkinson. 1977. A new type of penicillin resistance of Staphylococcus aureus. Lancet 1:443-447. 18. Schwarz, U., A. Asmus, and H. Frank. 1969. Autolytic enzymes and cell division of Escherichia coli. J. Mol.
963
Biol. 41:419-429. 19. Schwarz, U., and W. Weidel. 1965. Zum Wirkungpmechanismus von Penicillin. Z. Naturforsch. 206:147-157. 20. Spratt, B. G. 1975. Distinct penicillin binding proteins involved in the division, elongation and shape of Escherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A. 72: 2999-3003. 21. Spratt, B. G. 1977. Properties of the penicillin-binding proteins of Escherichia coli K-12. Eur. J. Biochem. 72: 341-352. 22. Suzuki, H., Y. Nishimura, and Y. Eirota. 1978. On the process of cellular division in Escherichia coli: a series of mutants of E. coli altered in the penicillin-binding proteins. Proc. Natl. Acad. Sci. U.S.A. 75:664-668. 23. Tamaki, S., S. Nakajima, and M. Matsuhashi. 1977. Thermosensitive mutation in Escherichia coli simultaneously causing defects in penicillin-binding protein lb-s and in enzyme activity for peptidoglycan synthesis in vitro. Proc. Natl. Acad. Sci. U.S.A. 74:5472-5476. 24. Tomasz, A. 1979. The mechanism of the irreversible antimicrobial effects of penicillins: how the beta lactam antibiotics kill and lyse bacteria. Annu. Rev. Microbiol. 33:113-137. 25. Tomasz, A., A. Albino, and E. Zanati. 1970. Multiple antibiotic resistance in a bacterium with suppressed autolytic system. Nature (London) 227:138-140. 26. Tomasz, A., and S. Waks. 1975. Mechanism of action of penicillin: triggering of the pneumococcal autolytic system. Proc. Natl. Acad. Sci. U.S.A. 72:4162-4166.
JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 955-963
0021-9193/79/12-0955/09$02.00/0
Escherichia coli Mutants Tolerant to Beta-Lactam Antibiotics KAZUAKI KITANO AND ALEXANDER TOMASZ* The Rockefeller University, New York, New York 10021 Received for publication 20 July 1979
Two types of Escherichia coli mutants tolerant to beta-lactam antibiotics were isolated. One is E. coli X2452, which showed a tolerant response against betalactam antibiotics when grown at 420C, and the others are the mutants C-80 and C-254, selected from mutagenized E. coli X 1776 by cycles of exposure to ampicillin, cephaloridine, and starvation of the nutritionally required diaminopimelic acid. Beta-lactam antibiotics caused rapid loss of viability and lysis in cultures of X1776 or in X2452 grown at 320C. In contrast, the same antibiotics caused only a reversible inhibition of growth in mutants C-80 and C-254 or in cultures of X2452 grown at 420C. Beta-lactam antibiotics that show high affinity for penicillinbinding proteins 2 or 3 (mecilhinam and cephalexin, respectively) induced similar morphological effects (ovoid cell formation and filament formation) in both parent and mutant strains. In contrast, beta-lactam antibiotics which have a high affinity for penicillin-binding protein 1 (e.g., cephaloridine or cefoxitin), which cause rapid lysis in the parental strains, caused cell elongation in the tolerant bacteria. In contrast to the parental cells, autolytic cell wall degradation was not triggered by beta-lactam treatment of X2452 cells grown at 420C or in mutants C-80 and C254. The total autolytic activity of mutants C-80 and C-254 was less than 30% that of the parent strain. However, virtually identical autolytic activities were found in cells of X2452 grown either at 42 or 320C. Possible mechanisms for the penicillin tolerance of E. coli are considered on the basis of these findings. Several types of biochemical observations suggest that lysis of Escherichia coli by beta-lactam antibiotics involves the activity of autolytic enzymes (5, 18, 19). Evidence for such an involvement exists in pneumococci since mutants selected for a defective autolytic system were found to be resistant to the lytic (and, to some degree, to the bactericidal) effect of beta-lactam antibiotics, while remaining sensitive to the growth inhibitory effect of these antibiotics (25, 26). Repeated attempts to select for autolysindefective E. coli have failed so far. Therefore, we decided to select for penicillin-tolerant mutants of E. coli, i.e., bacteria that could survive treatment with penicillin in a manner that is characteristic of the penicillin-tolerant pneumococci. We describe here three tolerant E. coli isolates. Each mutant was found to have a defective autolytic system. We chose the E. coli K-12 strain X1776 as the parental strain in the mutant isolation because of the presence of several useful properties of which of particular importance were the requirement for diaminopimelic acid (DAP), defective outer membrane, and sensitivity to detergentinduced lysis (4).
Although a complete characterization of the genetic and biochemical basis of tolerance in these bacteria is not yet available, it was felt that the existence of the first penicillin-tolerant, gram-negative mutants was of sufficient interest to warrant a preliminary description. MATERIALS AND MErTODS Bacterial strains and culture conditions. E. coli X2452 F- dapD AlacZ39 A(galbchl) A- tyrT58 naLA29 AthyA57 IyaA32 endAl asd hsdS3 (4), E. coli X1776
F- tonA53 dapD8 minAl supE42 A(gal-uvrB)40 AminB2 rfb-2 nalA25 oms-2 thyA57 metC65 oms-1 A(bioH-asd)29 cycB2 cycAl hsdR2, and E. coli C-80 and C-254 (beta-lactam tolerant mutants derived form X1776) were used. Because of the known mechanical fragility of both strains X1776 and x2452, bacteria were cultured without aeration. Cultures of 10 ml each were grown in test tubes (18 by 150 mm) at 32 or 420C in Penassay broth (Difco antibiotic medium no. 3) supplemented with DAP (20 ,ug/ml), L-lysine (100 yig/ml), biotin (0.2 ,ug/ml), and thymidine (30,ug/ml) (complete medium, OM). Cells radioactively labeled in the cell wall with DAP were obtained by growth in medium in which the DAP was replaced with 1 uCi of [3H]DAP per ml (giving a final DAP concentration of 3.1 ,ug/ ml). Isolation of mutants. E. coli X1776 was mutagen-
955
956
KITANO AND TOMASZ
ized by conventional procedures (1). Bacteria, grown exponentially at 320C in complete medium, were suspended in 0.05 M Tris-maleate buffer (pH 6.0) containing 300 ,ug of N-methyl-N'-nitro-N-nitrosoguanidine and were incubated for 30 min at 37°C. Celis were washed once with Tris-maleate buffer to remove the mutagen and suspended in OM. Portions of 2 ml were inoculated into 8 ml of OM and incubated at 320C for 3.5 h (cell concentration at this point was 5 x 107 to 7 x 107 cells per ml). Mutants were selected next by a cycle of treatments that included the following steps. Step (i). A 10-ml mutagenized culture was treated with ampicillin (20 ug/ml, corresponding to about five times the minimal inhibitory concentration [MIC]) for 1.2 h. During this treatment, the turbidity of the bacterial culture first increased by about 30% and then dropped to about 50% of the maximum turbidity value. The cells were collected by centrifugation (5,000 x g, 10 min, room temperature), washed once with OM, and resuspended in fresh OM (twice the original volume). After overnight incubation (13 h) at 320C, the surviving cells grew to a turbid culture that was resuspended in fresh OM and allowed to double in its turbidity (5 x 107 to 10 x 107 per ml), at which time a 10-ml portion of the culture received step (ii) of the enrichment procedure. Step (ii). Step (ii) consisted of treatment with cephaloridine (20 jug/ml; five times the MIC value) at 320C for 1.5 h. The turbidity changes of this culture were about the same as those observed during the first ampicillin treatment. After the cells were washed with OM, dilution and overnight incubation were the same as after ampicillin treatment. The overnight culture was washed with OM without DAP, resuspended in OM without DAP, and incubated at 320C for 3 h. Step (iii). During this DAP starvation there was an initial 20% increase followed by a decline (about 30% of the maximum) in turbidity. The culture was washed and resuspended in OM (to half of the culture's turbidity at the end of the DAP starvation) and incubated (320C) for 3 h, during which time the turbidity doubled. Portions of 10 ml of this culture were then exposed to the final step. Step (iv). Step (iv) consisted of ampicillin treatment (20 ,g/ml for 2.5 h). The bacteria were finally washed with OM and resuspended in 10 ml of OM, and frozen stocks were prepared (freezing at -40°C and storage at -80°C). The final scoring of mutants was done in the following way: frozen stock cultures were melted, diluted, and plated on OM agar plates (about 107 bacteria per plate) containing 10 Ag of ampicillin per ml and incubated at 320C for 16 h. Then the plates were overlayered with 4 ml of soft agar medium (0.6% agar in OM medium) containing 10 U of penicillinase (Rikers Chemicals) per ml and incubated for another 24 h. The colonies that grew up under this condition (about 100 per plate) were picked into liquid medium and checked for a tolerant response against beta-lactam antibiotics. Out of 340 such surviving colonies, 6 were found to show a substantially decreased rate of loss of turbidity (as compared with the parental culture) when challenged with cephaloridine (five times the MIC). These cultures also showed lower autolytic activity (30 to 60% of the parental cells). Two of these
J. BACTERIOL.
tolerant mutants, C-80 and C-254, exhibiting virtually no lysis during cephaloridine treatment, were chosen for more detailed characterization to be described in this paper. Assay procedures. Bacteria were routinely grown at 320C except when noted otherwise. Culture growth and culture lysis were monitored with a Coleman Nepho-colorimeter (14). Viable titers of the cultures were assayed by routine plating procedures. The assay method of triggered autolysin by beta-lactam antibiotics (13) was as follows. After growth in medium supplemented with [3H]DAP for several generations, cells in the exponential growth phase were collected by centrifugation (4,300 x g, 5 min), transferred to radioactive isotope-free growth medium, and incubated for 50 min (i.e., a period of about one generation) to deplete cellular pools of the [3H]DAP. After this period, 1.5-ml portions of the culture were distributed into a number of small tubes containing beta-lactam antibiotics at various concentrations (representing multiples of the corresponding MICs) and incubated for an additional 10 to 20 min. The cultures were chilled (ice bath) and then centrifuged at 3,300 x g for 5 min at 40C. Cells were washed with 1.5 ml of ice-cold phosphate buffer (0.1 M, pH 7.0), resuspended in 1.5 ml of the same buffer containing 10 mM MgSO4, and incubated at 320C. The total time needed to transfer the cells to the buffer was about 10 to 15 min. After 0, 30, 60, 90, and 120 min of incubation, 200-p1 portions were removed into prechilled Eppendorf microcentrifuge tubes containing 20 pl of 38% formaldehyde (to stop murein hydrolase activity). After centrifugation at 12,000 x g for 10 min in the cold (40C), radioactivity in 100 pl of the supernatant fluids was counted. To determine total radioactivity of the reaction mixture, 200-pd portions were mixed with 20 p1 of 4% deoxycholate and incubated for 30 min at 320C; a 100-pl portion of this suspension was used to determine radioactivity. The activity of autolysin triggered by beta-lactam antibiotics was expressed as the rate of degradation of murein during a 2-h incubation of the beta-lactam antibiotic-treated cells in buffer. The rates were corrected for the spontaneous rate of release of radioactivity from the control (untreated) cells. The treatment time with beta-lactam antibiotics was 20 min (for E. coli X2452 cells) or 10 min (for the cells of E. coli x1776 C-80 and C-254). Triggering of murein hydrolase by trichloroacetic acid. The method of Schwarz et al. was used (18) (see also legend to Fig. 8) to trigger murein hydrolase by trichloroacetic acid. Reagents. DL-(meso)-2,6-DiamiIno-[U-3H]pimelic acid (1.5 Ci/mmol) was purchased from Amersham Corp. (Arlington Heights, Ill.). All other materials and chemicals were reagent-grade commercially available products.
RESULTS Tolerant response of E. coli X2462 to betalactam antibiotics at 420C. Several mutants of E. coli K-12 were examined for their response to beta-lactam antibiotics and a mutant, E. coli X2452, was found to show a tolerant response to
BETA-LACTAM-TOLERANT MUTANTS OF E. COLI
VOL. 140, 1979
beta-lactams at 42°C but not at 32°C (Fig. 1). X2452 was isolated by Dennis Pereira in the laboratory of Roy Curtiss (D. Pereira, Ph.D. Thesis, University of Alabama Medical Center, Birmingham, Ala., 1979). Although rapid culture lysis occurred after exposure to the MIC of cephaloridine at 320C, the cells were stable to lysis by the addition of cephaloridine when the bacteria were grown at 420C. This phenomenon was reproducible by the addition of other betalactams such as ampicillin, benzylpenicillin, or cephalothin. The MICs of these beta-lactams were identical at the two temperatures (Table 1). The tolerant response to beta-lactam antibiotics was temperature dependent, and if temperature was shifted down from 42 to 320C at 0 to 30 min after the addition of the drug, cells started lysing after a short time lag (Fig. 2). Tolerant response of E. coli C-80 and C254 to beta-lactam antibiotics. Figure 3 demonstrates the effect of cephaloridine on cultures of E. coli X1776, C-80, and C-254. Although growth of the mutants was stopped by the addition of cephaloridine, the cells scarcely lysed even after prolonged (overnight) incubation with the antibiotic. Culture lysis was very slow or negligible in all of the tolerant mutants (including X2452 grown at 4200) even during exposure
957
TABLE 1. MICs of beta-lactam antibiotics against mutants of E. colia MIC (jug/ml) of strain: Antibiotic
X2452
X1776
C-80
C-254
320C 420C 3.9 3.9 3.9 3.9 15.6 15.6 31.25 31.25
Cephaloridine 2.0 3.9 3.9 Ampicillin 3.9 3.9 3.9 Cephalothin 31.25 62.5 62.5 Benzylpenicillin 31.25 62.5 62.5 Cefoxitin 7.8 7.8 7.8 Cephalexin 15.6 Mecillinam 7.8 7.8 7.8 aThe MICs listed in the table were determined by the following procedure: exponentially growing cultures (at cell concentration of about 5 x 107 cells per ml) received antibiotics at various concentrations, and growth of the cultures was monitored (as described in the Materials and Methods). The minimum antibiotic concentration that caused cessation of growth was taken as the MIC. Inhibition of growth was not instant but became manifest only after a residual increase in the turbidity of cultures.
400t
N 200}
42 C 100'
50'
0
1
2
HOURS FIG. 2. Effect of temperature shift during treatment of E. coli X2452 with cephaloridine. Bacterial cultures were grown in OM at 42°C without aeration. At a cell concentration of about 1 x io0 viable cells per ml (in the late exponential phase of growth) (arrow), the cultures received 2.5 times the MIC of cephaloridine (19.5 pg/ml), and the temperature of the cultures was shifted to 320C at 0 (A), 15 (V), and 0 1 2 3 0 1 2 3 30 (El) min after the addition of the drug. Control HOURS cultures at 42°C with (O) or without (*) cephaloriFIG. 1. Effect of cephaloridine on cultures of E. dine are also shown. coli X2452 growing at 32 and 42°C. Bacterial cultures were grown in OM without aeraition. In the late to high concentrations (eight times the MIC) of exponential phase of growth (arrrow), the cultures and a variety of other cephaloridine, received cephaloridine at the conc ples of MIC; 7.8 pg/ml at both te,mperatures) indi beta-lactam antibiotics. Nevertheless, inhibitors cated by the numbers, and the grou pth response of the of early stages of cell wall synthesis (e.g., the bacterial cultures was followed by inephelometry and combination of D-cycloserine and fluoro-D-alaplotted in nephelometric (N) units. nine) could still induce lysis of the tolerant bac-
ampicillin,
958
KITANO AND TOMASZ
J. BACTERIOL.
X-1 776
C-254
C -80
400
N
1004 so
0
1
2
3
0 1 2 3 0 1 2 3 HOURS FIG. 3. Effect of cephaloridine on cultures of E. coli X1776 and its mutants C-80 and C-254. Bacterial
cultures were grown in OM at 32°C without aeration. In the exponential phase ofgrowth (5 x 107 viable cells per ml) (arrow), the cultures received antibiotics at the concentrations (multiples of MIC) indicated by the numbers, and the growth response of the bacterial cultures was followed by nephelometry. The MICs for cephaloridine were as follows: xl776, 3.9 pg/ml; mutants C254 and C-80, 7.8 pg/ml.
teria (data not shown). The MICs of various beta-lactams (for method of MIC determination see footnote a, Table 1) were identical to those of the parental cells (Table 1) except for benzylpenicilhin and cephalothin; in these cases the MIC for the tolerant cells was higher (maximum, twice those of the parents). Cultivation of E. coli X2452 at 420C gave a striking protection against the killing effects of cephaloridine (Fig. 4a) and also against lysis. The killing effects of cephaloridine in the mutants C-80 and C-254 were also very weak as compared with the effects in the parental strain (Fig. 4b). Morphological changes of the mutant cells during treatment with beta-lactams. Figure 5 demonstrates the effect of three betalactam antibiotics on the cell shape of E. coli X2452 at 42 and 320C. Cephaloridine, which has a high affinity for penicillin-binding protein (PBP) lb (20, 21) caused rapid lysis of cells at 320C and the formation of empty cells, whereas at the nonpermissive temperature for lysis, cell elongation continued, and bacilli became noticeably longer than the control cells. Mecillinam (which has a high affinity for PBP 2 [20]) caused the formation of ovoid cells at both 32 and 420C. During the 3 h of treatment with cephalexin, cells grown at 420C have elongated to lengths varying between 1.5 and 3 times the length of the untreated bacteria grown at this temperature. The same drug caused a more substantial elongation (3 to 6 times the normal length) when
CX-254
x
C°~X-1776
4._ co
B 2
1
2
HOURS FIG. 4. Bactericidal action of cephaloridine against beta-lactam-tolerant mutants of E. coli. (A) E. coli X2452 cells growing in complete medium at 32 and 42°C were treated at 0 min (cell concentration, 1 x 108 bacteria per ml) with 20 pg (about 2.5 times the MIC) of cephaloridine per ml. (B) Cells of E. coli X1776 C-80 and C-254 growing in complete medium at 320C were treated at 0 min with twice the MIC of cephaloridine. Samples were periodically removed, diluted with complete medium, and plated to determine the number of viable cells.
the cells were grown at 320C. Similar phenomena were also observed with the tolerant mutants C-80 (Fig. 6) and C-254 (data not shown). In contrast, the mecillinam-induced ovoid cell formation was not easy to document in these tolerant strains or in the parental strain X1776. Mecillinam treatment, at least under the conditions used here (i.e., relatively high cell concen-
VOL. 140, 1979
vi. .i,j4
~*
959
BETA-LACTAM-TOLERANT MUTANTS OF E. COLI -4
........ . . t~ ~ ~ ~ ~ ~ ~ ~ . .
.jt
*$,. .. . b
I! \
:~~~~~ *_
A.
(
B~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~:
4~~. 2.B i-
1A¢.-: . x At; .:....''
v
4
.
S'M |
..
/
:0
. ,f '
.d'':
.I
dp
I
/_....
IF
^
6
I
_1 lb
I-
_a
IN
.FIG. 5. Effect of beta-lactam antibiotics on cell shape of E. coli X2452 grown at 32 and 42°C. (A) Control; (B) cephaloridine; (C) mecillinam; (D) cephalexin. Cells grown at 32 and 42°C were exposed to four times the MICs of various beta-lactam antibiotics for 3 h. Cells were fixed with 2% glutaraldehyde, and phase-contrast micrographs were then made of the cells, using a Zeiss microscope fitted with a Planachromat 100/1.25 phasecontrast oil immersion objective. Kodak Panatomic-X film was used for the photomicrography.
tration), caused the formation of abnormally shaped bacteria (Fig. 6). Triggering of autolytic activity by cephaloridine and by trichloroacetic acid treatment. The autolytic cell wall degradation of E. coli X2452 was effectively triggered by brief exposure (10 to 20 min) to beta-lactam antibiotics at the lysis-permissive temperature but was not triggered at 42°C (Fig. 7). Autolysis of mutants C-80 and C-254 was not triggered by this treatment either (Fig. 7). Interestingly, exposure to high concentrations of antibiotics (eight times the MIC [Fig. 7]) has occasionally caused a suppression of the rate of cell wall degradation. This type of effect has been consistently observed at very high concentrations (10 to 100 times the MICs) of certain beta-lactam antibiotics (13). The autolytic activity of E. coli X2452 triggered by 5% trichloroacetic acid treatment (7) was almost the same in cells grown either at 32 or 420C and was independent of the temperature at which the assays were carried out (Fig. 8a). In contrast, the autolytic activities of C-80 and C-254 triggered by 5% trichloroacetic acid treatment were less than 30% that of the parent strain
(Fig. 8b). Identical results were obtained when autolysis was triggered by other methods (exposure to hypertonic sucrose or to freezing and thawing [7]) or in experiments in which the autolytic activities of Triton-ethylenediaminetetraacetic acid extracts (7) (made from tolerant and lysis-prone cells) were compared. DISCUSSION The mechanisms by which inhibition of penicillin-sensitive bacterial enzymes (or PBP) brings about interference with the growth of a bacterial cell is not clearly understood presently (24). Chemically different beta-lactam antibiotics can elicit a variety of different morphological effects and distinct growth inhibitory mechanisms in the same bacterium (E. coli) (20, 21). There are also examples of the converse situation since different species of bacteria treated with the same beta-lactam antibiotic may show a wide variety of physiological responses, such as inhibition of growth (with only an extremely slow loss of viability), rapid loss of viability, and loss of viability accompanied by cellular lysis (9, 26). Studies on pneumococci and some other gram-positive bacteria have shown that the
960
KITANO AND TOMASZ
X -17 76
;
|
J. BACTERIOL.
_
........
000, .:....::.
C-80
4koolo
q*4
0 4e : :. .,.1
a 0
I
Ls
A,
I
0
/
v
1.
FIG. 6. Effect of beta-lactam antibiotics on cell shape of E. coli X1776 and its mutants. (A) Control; (B) cephaloridine; (C) mecillinam; (D) cephalexin. Cultures of the bacteria grown at 32°C were exposed to four times the MICs of the various beta-lactam antibiotics for 3 h. After fixation with 2% glutaraldehyde, phasecontrast micrographs were made of the bacteria, using a Zeiss microscope fitted with a Planachromat 100/ 1.25 phase-contrast oil immersion objective. Kodak Panatomic-X filn was used for the photomicrography.
beta-lactam antibiotic-induced lysis requires the activity of bacterial autolysins (2, 6, 26). Pneumococci in which the in vivo activity of the Nacetyl muramic acid-L-alanine amidase has been suppressed (by mutation or physiological manipulations) show a unique response to penicillin treatment: in such cells the antibiotic causes inhibition of growth (at the normal MIC) and often the rate of loss of viability is also greatly reduced, but no lysis occurs (25, 26). We proposed the term "antibiotic tolerance" for this phenomenon (25), and recent reports suggest that antibiotic tolerance may not be restricted to laboratory strains of pneumococci but may also occur among clinical isolates of Staphylococcus aureus (17) and Streptococcus sanguis (10). The involvement of autolytic activity in the penicillin-induced lysis of E. coli has been repeatedly suggested in the literature, and penicillin treatment of E. coli has been reported to cause enzymatic cell wall degradation (19) and also an increase in the in situ autolytic activity (5). In a recent, more detailed study, we found a striking quantitative correlation between the efficiency of beta-lactam antibiotics to trigger autolytic cell wall degradation and the relative affinity of these antibiotics for the PBP 1 group of E. coli (13). In this com-
munication we describe the properties of three E. coli isolates that exhibit a tolerant response to treatment with penicillins and cephalosporins. Each one of the isolates has a defective autolytic system. One of the isolates (x2452) exhibits a tolerant response when grown at 420C and a lytic bactericidal response when grown at 32°C. The other two mutants (C-80 and C-254) show tolerance at all temperatures of growth. These E. coli mutants resemble the autolysis-defective pneumococci in that each one of them exhibits an abnormality in their autolytic system: brief treatment of the mutants (or of X2452 grown at 4200) with beta-lactam antibiotics does not trigger cell wall degradation, although the same treatment causes rapid and extensive wall hydrolysis in the parental bacteria and in strain X2452 grown at the lysis-permissive temperature (320C). Additional tests have revealed a substantially decreased autolytic activity in the mutant cells (as compared with the parental bacteria). However, cells of strain X2452 grown at either 32 or 420C appear to have comparable (normal) levels of autolysin activity. Thus, tolerance of mutants C80 and C-254 is accompanied by an apparent net decrease in autolysin activity, whereas in X2452 only the triggering of autolytic activity seems
BETA-LACTAM-TOLERANT MUTANTS OF E. COLI
VOL. 140, 1979
961
The mechanism by which interference with cell wall synthesis provokes autolytic cell wall 32 20D degradation in E. coli is not yet known. In the .0 0 case of pneumococci, we proposed that the suia2.0~ cidal activity of the autolytic amidase may be caused by a defect that rapidly upsets the in control of this enzyme after the addition of vivo , 1.5 to the bacteria (26). This suggestion penicillin 0 on the demonstrated release of an was based 30 autolysin inhibitory agent (Forssman antigen) ._ 31 42 C 1.0D from pneumococci during penicillin treatment (26). There is no evidence at this time for the existence of analogous autolysin inhibitors in E. 2 4 8 1 coli. The triggering of in situ autolytic wall degAntibiotic concentration (MIC units) radation of E. coli after exposure of the cells to FIG. 7. Triggering of autolytic activity by cepha- various chemical treatments (e.g., cold trichloloridine. After growth in medium supplemented with roacetic acid, ethylenediaminetetraacetic acid, [3H]DAP for several generations, cells were grown in radioisotope-free medium for an additional cell gen- high concentration of sucrose [7]) or mechanical eration. After this period, 1.5-ml portions of the cul- disruption has been explained as the disruption ture were treated with increasing concentrations of of a "barrier" (plasma membrane?) that was cephaloridine for 20 min (strain x2452) or for 10 min suggested to separate the autolysins of E. coli (strain xl776 and its mutant derivatives). The growth from their substrate in the normal cells (7). temperature was 320C for all strains except for a Rapid release of substantial quantities of lipids portion of X2452, which was also grown at the lysis- and other membrane components have been protective temperature of 42°C. For the assay of cell demonstrated during penicillin treatment of sevwall degradation, cells of all strains (including the 420C-grown bacteria) were incubated at 32°C after eral autolysin-defective gram-positive bacteria washing and resuspension in buffer (1.5 ml). Radio- (9,11, 12). Preliminary experiments indicate that activity released during 2 h of incubation in the buffer penicillin-induced release of lipid material also occurs in some strains of E. coli (A. Tomasz, was counted, and the autolytic activity was expressed unpublished data), and it is conceivable that as the rate relative to the spontaneous rate of release of radioactivity from the untreated bacteria. This such a process may damage the hypothetical X-2452
C
,,, ;
latter value has amounted to about 8% of the total incorporated radioactivity (13).
thermosensitive. The titration of the total autolysin content of E. coli presents considerable problems since there are several enzyme activities involved (8, 16) and some of these are structure bound. Furthermore, in extracts, some of these hydrolytic enzymes may be restricted in their ability to attack cell walls. For these reasons, it was felt that a determination of in situ autolysin activity (7) as it was done in our experiments may be an appropriate first approximation when comparing autolysin levels of tolerant (mutant) and lysis-prone (parental) bacteria. The level of autolysin activity was also determined in Triton-EDTA extracts (7) prepared from the parental cells and from the mutants C80 and C-254. The mutant extracts showed a diminished specific hydrolase activity (about 25 to 30% that of the extract from parent cells). In these assays radioactive DAP-labeled cell walls (murein sacculi [3]) from the parental bacteria were used as substrate (data not shown). It is not yet known whether the lowered autolysin activity in the mutants involves one or more of the multiple types of murein hydrolases of E. coli (8, 16).
60
A
B
.C 50
E 42
X-1776 ,
/
0 3 *
0
;
32 C C ~~~~~~42
.'20
C-254
=>1> ,,
1
,
2
;AtS--~~~~~80 1
2
HOURS FIG. 8. Autolytic activity triggered by trichloroacetic acid treatment. [3H]DAP-labeled cells were suspended in 0.01 M Tris-maleate buffer (pH 6.0) containing 10 mM MgSO4 and mixed with an equal volume of 10% trichloroacetic acid and allowed to react for 10 min in ice (7). The cells were washed three times by centrifugation in the buffer, resuspended in the same buffer, and incubated at 32°C. The radioactivity released into the supernatant solution was assayed at different times by theprocedure described in Materials and Methods. (A) Cells of X2452 grown at 32°C (O) or 420C (*). (B) Cells of X1776 (O) and its mutants C-80 (*) and C-254 (A). Bacteria were grown at 320C.
962
KITANO AND TOMASZ
barrier separating autolysins and the murein in a manner somewhat analogous to the damage that may be responsible for triggering by chemical treatments. If this were true, then the lack of autolysin triggering in strain X2452 grown at 420C may be caused by the presence of a more stable barrier (different membrane composition). Alternatively, cell wall synthesis at the higher temperature may produce a murein of low autolysin sensitivity at a critical area of the mutant cell wall. It should be emphasized that the nature of autolysin triggering is obscure at the present time. There is strong evidence implicating the functional involvement of both the binding proteins 1 (PBP la and lb) (21-23) as well as the autolytic enzyme(s) of E. coli in the penicillininduced lysis of this bacterium. However, it is not clear how and why inhibition of PBP leads to initiation of autolytic cell wall degradation, and the term triggering refers to this poorly understood connection between the functions of PBP 1 and the control of autolysin activity. Besides the hypothetical barrier function, triggering may involve accumulation of cell wall precursors or even the local modification of the murein substrate, e.g., by introduction of poorly cross-linked material into areas of the cell wall during penicillin treatment (15). In the thermosensitive tolerant mutant X2452 the heat-sensitive element in the penicillin response seems to be this hypothetical triggering process since both autolytic activity and the PBP patterns are normal at each temperature (data not shown). The substantially lowered autolytic activity and antibiotic tolerance of mutants C-80 and C-254 are reminiscent of the properties of autolysis-defective pneumococci. On the other hand, lysis of the E. coli mutants can still be induced by treatment with inhibitors of early steps in cell wall biosynthesis, whereas the autolysis defective pneumococci are resistant to the lytic effect of all cell wall inhibitors (25). It is conceivable that the tolerant phenotype has a more complex biochemical and genetic basis in E. coli than in pneumococci because of the more complex nature of autolysin regulation and the differences in the mode of cell wall assembly in E. coli and in pneumococci. The morphological changes induced in the tolerant E. coli during treatment with different beta-lactam antibiotics require comment. It seems that inhibitors of PBP 2 (mecillinam) and PBP 3 (cephalexin) can elicit their typical morphological effects in these cells (i.e., ovoid cell formation and filament formation), whereas the response of the tolerant E. coli to inhibitors of PBP 1 is changed: instead of the appearance of
J. BACTERIOL.
"rabbit ear" forms and lysis, such cells would elongate (Fig. 5). These findings support the notion that in E. coli triggering of autolysin activity by beta-lactam antibiotics is a specific consequence of the inhibition of PBP 1. The tolerant cells appear to be defective in the sequence of events leading from PBP 1 to autolysin triggering; they respond to inhibitors of PBP 1 by reversible inhibition of growth (instead of loss of viability and lysis). These observations suggest that triggered autolysin activity may be the cause of viability loss (as well as cell lysis) in E. coli. It is hoped that these mutants will be helpful in the analysis of the mechanism of the antimicrobial effect of beta-lactam antibiotics. A mutant of E. coli strain K-12 that is tolerant to lysis by cephalexin has been isolated independently by T. Nikaido, S. Tomioka, and M. Matsuhashi (personal communication). No defect in a peptidoglycan-lytic enzyme activity has so far been demonstrated. ACKNOMWLEDGMENTS These investigations have been supported by grants from the National Science Foundation (PCM 7812770) and the National Institutes of Health (AI 12932). We thank Roy Curtiss III and Dennis Pereira (University of Alabama, Birmingham, Ala.) for providing us with strains X1776 and X2452
LITERATURE CITED 1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. Optimal conditions for mutagenesis by N-methyl-N'nitro-N-nitrosoguanidine in Escherichia coli. Biochem. Biophys. Res. Commun. 18:788-795. 2. Ayusawa, D., Y. Yoneda, K. Yamane, and B. Maruo. 1975. Pleiotropic phenomena in autolytic enzyme(s) content, flagellation, and simultaneous hyperproduction of extracellular a-amylase and protease in a BaciUus subtilis mutant. J. Bacteriol. 124:459-469. 3. Braun, V., and K. Rehn. 1969. Chemical characterization, spatial distribution and function of a lipoprotein (murein-lipoprotein) of the E. coli cell wall: the specific effect of trypsin on the membrane structure. Eur. J. Biochem. 10:426-438. 4. Curtiss, R. m. 1978. Biological containment and cloning vector transmissibility. J. Infect. Dis. 137:668-675. 5. Fontana, R., G. Satta, and C. A. Romanzi. 1977. Penicillins activate autolysins extracted from both Escherichia coli and Kkbsiella pneumoniae envelopes. Antimicrob. Agents Chemother. 12:745-747. 6. Forsberg, C., and H. J. Rogers. 1971. Autolytic enzymes in growth of bacteria. Nature (London) 229:272-273. 7. Hartmann, R., B. S. Bock-Henning, and U. Schwarz. 1974. Murein hydrolases in the envelope of Escherichia coli. Eur. J. Biochem. 41:203-208. 8. Holtje, J. V., D. Mirelman, N. Sharon, and U. Schwarz. 1975. Novel type of murein transglycosylase in Escherichia coli. J. Bacteriol. 124:1067-1076. 9. Horne, D., R. Hakenbeck, and A. Tomasz. 1977. Secretion of lipids induced by inhibition of peptidoglycan synthesis in streptococci. J. Bacteriol. 132:704-717. 10. Horne, D., and A. Tomasz. 1977. Tolerant response of
VOL. 140, 1979
BETA-LACTAM-TOLERANT MUTANTS OF E. COLI
Streptococcus sanguis to beta-lactams and other cell wall inhibitors. Antimicrob. Agents Chemother. 11: 888-896. 11. Horne, D., and A. Tomasz. 1979. Release of lipoteichoic acid from Streptococcus sanguis: stimulation of release during penicillin treatment. J. Bacteriol. 137:11801184. 12. Kikuchi, M., T. Kanamaru, and Y. Nakao. 1973. Relation between the extracellular accumulation of L-glutamic acid and the excretion of phospholipids by penicillin-treated Corynebacterium alkanolyticum. Agr. Biol. Chem. 37:2405-2408. 13. Kitano, K., and A. Tomasz. 1979. Triggering of autolytic cell wall degradation in Escherichia coli by beta-lactam antibiotics. Antimicrob. Agents Chemother. 16:838848. 14. Meynell, G. G., and E. Meynell. 1970. Theory and practice in experimental bacteriology. Cambridge University Press, Cambridge. 15. Oka, T. 1976. Mode of action of penicillins in vivo and in vitro in Bacillus megaterium. Antimicrob. Agents Chemother. 10:579-591. 16. Pelzer, H. 1963. Mucopeptidhydrolasen in Escherichia coli 13 I. Nachweis und Wirkungspecifitat. Z. Naturforsch. 18B:950-956. 17. Sabath, L. D., N. Wheeler, M. Laverdiere, D. Blazevic, and B. Wilkinson. 1977. A new type of penicillin resistance of Staphylococcus aureus. Lancet 1:443-447. 18. Schwarz, U., A. Asmus, and H. Frank. 1969. Autolytic enzymes and cell division of Escherichia coli. J. Mol.
963
Biol. 41:419-429. 19. Schwarz, U., and W. Weidel. 1965. Zum Wirkungpmechanismus von Penicillin. Z. Naturforsch. 206:147-157. 20. Spratt, B. G. 1975. Distinct penicillin binding proteins involved in the division, elongation and shape of Escherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A. 72: 2999-3003. 21. Spratt, B. G. 1977. Properties of the penicillin-binding proteins of Escherichia coli K-12. Eur. J. Biochem. 72: 341-352. 22. Suzuki, H., Y. Nishimura, and Y. Eirota. 1978. On the process of cellular division in Escherichia coli: a series of mutants of E. coli altered in the penicillin-binding proteins. Proc. Natl. Acad. Sci. U.S.A. 75:664-668. 23. Tamaki, S., S. Nakajima, and M. Matsuhashi. 1977. Thermosensitive mutation in Escherichia coli simultaneously causing defects in penicillin-binding protein lb-s and in enzyme activity for peptidoglycan synthesis in vitro. Proc. Natl. Acad. Sci. U.S.A. 74:5472-5476. 24. Tomasz, A. 1979. The mechanism of the irreversible antimicrobial effects of penicillins: how the beta lactam antibiotics kill and lyse bacteria. Annu. Rev. Microbiol. 33:113-137. 25. Tomasz, A., A. Albino, and E. Zanati. 1970. Multiple antibiotic resistance in a bacterium with suppressed autolytic system. Nature (London) 227:138-140. 26. Tomasz, A., and S. Waks. 1975. Mechanism of action of penicillin: triggering of the pneumococcal autolytic system. Proc. Natl. Acad. Sci. U.S.A. 72:4162-4166.
